双黄连制剂中酚酸与黄酮类成分的含量测定及药代动力学分析

Determination and Pharmacokinetic Analysis of Phenolic Acids and Flavonoids After Oral Administration of Shuanghuanglian Preparation by UPLC-MS/MS

目的：建立同时测定大鼠灌服双黄连制剂后血浆中酚酸类（新绿原酸、绿原酸及隐绿原酸）与黄酮类（黄芩苷、千层纸素A-7-O-β-D-葡萄糖苷、汉黄芩苷、黄芩素、汉黄芩素及千层纸素A）成分的超高效液相色谱-串联质谱法（UPLC-MS/MS）,并研究其药代动力学。方法：血浆样品采用乙酸乙酯-正丁醇（1∶1）萃取,流动相0.4%甲酸水溶液-[乙腈-甲醇（4∶1）混合液]梯度洗脱,流速0.4 m L·min-1,采用电喷雾离子化,多反应监测离子扫描模式,选用响应较高的正离子模式。结果：建立的同时测定血浆中9种成分的UPLC-MS/MS线性良好（R2〉0.992）,精密度RSD均〈14.2%,准确度85.49%~114.76%,复方中新绿原酸、绿原酸、隐绿原酸、黄芩苷、千层纸素A-7-O-β-D-葡萄糖苷、汉黄芩苷、黄芩素的药时曲线下面积AUC0-t与AUC0-∞与单味药提取物相比显著增加,而酚酸和黄酮类成分的消除速率较单味药提取液缓慢。结论：建立的UPLC-MS/MS特异、快速、准确及灵敏,可用于双黄连制剂中9种主要活性成分在大鼠体内的药代动力学研究。

Objective： A rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method（ UPLC-MS / MS） was developed and validated for simultaneously determination of phenolic acids and flavonoids in rat plasma after oral administration of Shuanghuanglian preparation. Method： Plasma samples were pretreated by n-butanol-ethyl acetate（ 1 ∶ 1）. The separation was preformed on an Waters Acquity HSS T3 C18column（ 2. 1 mm × 100 mm,1. 7 μm） and the flow rate was set at 0. 4 m L·min- 1with mobile phase of acetonitrile-methanol（ 4 ∶ 1）-0. 4% formic acid for gradient elution. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring（ MRM） via electrospray ionization（ ESI）source with positive mode. Result： All calibration curves had good linearity（ R2 0. 992）. RSDs of precisionand accuracy were all satisfied. After oral administration of Shuanghuanglian preparation,a remarkable increase in values of AUC0-t and AUC0-∞of neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,baicalin,oroxylin A-7-O-β-D-glucuronide,wogonoside and baicalein was observed by compared with Lonicerae Japonicae Flos extract group or Scutellariae Radix extract group; but the elimination rate of phenolic acids and flavonoids in Shuanghuanglian preparation was slower than the single herb extract. Conclusion： The developed method is successfully applied to pharmacokinetic study of nine active ingredients in Shuanghuanglian preparation,this study can provide experimental evidence for elucidating rationality of compatibility of this preparation.